Apparatus and method of obtaining an image of a sample in motion

ABSTRACT

A method is used to generate an analysis image of a moving sample based on one or more exposures. An illumination source illuminates a field of view of a camera for one or more pulses while the sample moves through the field of view. The distance moved by the sample during each of these one or more pulses may be less than the size of one pixel in an image captured by the camera.

CROSS REFERENCE TO RELATED APPLICATIONS

This claims the benefit of U.S. provisional patent application63/110,720, entitled “Apparatus and Method of Obtaining an Image of aSample in Motion,” filed on Nov. 6, 2020, which is incorporated byreference herein in its entirety.

BACKGROUND

The subject matter discussed in this section should not be assumed to beprior art merely as a result of its mention in this section. Similarly,a problem mentioned in this section or associated with the subjectmatter provided as background should not be assumed to have beenpreviously recognized in the prior art. The subject matter in thissection merely represents different approaches, which in and ofthemselves may also correspond to implementations of the claimedtechnology.

For an object to be imaged, photons must be collected while that objectis in the field of view of an imaging device. This, in turn, requiresthe object to be illuminated. When the object to be imaged is only inthe field of view for a limited time, an imaging system must ensure thatthe energy applied through illumination during the time the object is inthe field of view is sufficient for the necessary photons to becollected. High precision motion stages, time delay integration (TDI)cameras, and diode pumped solid state (DPSS) lasers are among thecomponents that have been used to achieve this objective.

SUMMARY

Examples disclosed herein are directed to techniques for illumination ofobjects, and focuses particularly on techniques for illumination ofsamples of genetic material to be sequenced.

An implementation relates to a machine comprising a camera to captureimages comprising pixels, each of which has a pixel size correspondingto a distance on a stage in a direction of movement of a samplecontainer. The machine further comprises the stage to move the samplecontainer relative to a field of view of the camera which overlaps thestage, wherein the sample container comprises an array of featureshaving a pitch length in the direction of movement of the samplecontainer. The machine further comprises an illumination source toilluminate the field of view of the camera. The machine furthercomprises a controller to obtain an analysis image by performing actscomprising, while a feature from the array of features is in, and is inmotion relative to, the field of view of the camera, obtaining one ormore exposures of the feature. Obtaining one or more exposures of thefeature may be performed by, for each of the one or more exposures,performing acts. The acts may comprise exposing a sensor of the camerato illumination for a first duration and, during a period having asecond duration which takes place while the sensor of the camera isexposed to illumination, illuminating the field of view of the camerawith the illumination source. In such a machine, the feature'sdisplacement in the field of view of the camera from beginning to end ofthe period having the second duration is less than or equal to the pitchlength in the direction of movement of the sample container.

In some implementations, in a machine such as described in thepreceding, the feature's displacement in the direction of movement ofthe sample container in the field of view of the camera from beginningto end of the period having the second duration may be less than orequal to the pixel size.

In some implementations of a machine such as that described in either ofthe preceding two paragraphs of this summary, obtaining one or moreexposures of the feature comprises obtaining a plurality of exposures ofthe feature. The acts the controller is to perform comprise, overlayingthe plurality of exposures of the feature based on translating one ormore of the plurality of exposures of the feature.

In some implementations of a machine such as described in the precedingparagraph of this summary, the acts the controller is to performcomprise, for each exposure, obtaining a corresponding value for thesample container's position when the field of view of the camera wasilluminated with the illumination source. In some such implementations,the controller is to translate one or more of the plurality of exposuresof the feature based on differences between the exposures' correspondingvalues for the sample container's position.

In some implementations of a machine such as described in the precedingparagraph of this summary, the machine comprises an encoder to providevalues for the sample container's position. In some suchimplementations, the controller is to, for each exposure, obtain thecorresponding value for the sample container's position when the fieldof view of the camera was illuminated with the illumination source fromthe encoder.

In some implementations of a machine such as described in the precedingparagraph of this summary, the encoder has a resolution to distinguishdistances smaller than the distance on the stage corresponding to thepixel size and overlaying the plurality of exposures of the featurecomprises co-registering each of the plurality of exposures at theresolution of the encoder.

In some implementations of a machine such as described in the precedingparagraph of this summary, co-registering each of the plurality ofexposures at the resolution of the encoder comprises, for at least oneof the one or more exposures, obtaining a frequency space representationby taking a fast Fourier transform of the exposure. Co-registering eachof the plurality of exposures at the resolution of the encoder furthercomprises, for at least one of the one or more exposures, translatingthe frequency space representation by the distance which is not a wholenumber multiple of the distance on the stage corresponding to the pixelsize. Co-registering each of the plurality of exposures at theresolution of the encoder further comprises, for at least one of the oneor more exposures, performing an inverse fast Fourier transform of thetranslated frequency space representation.

In some implementations of a machine such as described in either of thepreceding two paragraphs of this summary, co-registering each of theplurality of exposures at the resolution of the encoder comprises, foreach of the plurality of exposures: upsampling that exposure to theresolution of the encoder based on interpolating data between pixels,and translating one or more of the exposures after upsampling.

In some implementations of a machine such as described in any of thepreceding paragraphs of this summary, the sample container may comprisea plurality of fiducial points, and the controller may be to translateone or more of the plurality of exposures of the features based ondifferences in location of the fiducial points between exposures.

In some implementations of a machine such as described in any of thepreceding paragraphs of this summary, the analysis image comprises aplurality of pixels, each having a first bit depth. In some suchimplementations, each of the plurality of exposures comprises aplurality of pixels, each of which has a second bit depth which is lessthan the first bit depth.

In some implementations of a machine such as described in the precedingparagraph, each pixel comprised by each image captured by the camera hasa third bit depth, wherein the third bit depth is greater than thesecond bit depth. Obtaining the plurality of exposures of the exposurecomprises, for each exposure, capturing an image with the camera whilethe field of view of the camera is illuminated by the illuminationsource; and truncating a number of most significant bits of the pixelsfrom the image captured by the camera, wherein the truncated number ofmost significant bits is equal to the difference between the third bitdepth and the second bit depth

In some implementations of a machine such as described in any of thepreceding paragraphs of this summary, a threshold illumination energydose is required for imaging the feature. For each of the one or moreexposures of the feature, illuminating the field of view of the camerawith the illumination source comprises activating the illuminationsource at a power which when multiplied by the second duration, providesan individual exposure energy dose less than the threshold illuminationenergy dose for imaging the feature, and when multiplied by the secondduration and multiplied by the number of exposures in the plurality ofexposures, provides a collective exposure energy dose greater than thethreshold illumination energy dose for imaging the feature.

In some implementations of a machine such as described in any of thepreceding paragraphs of this summary, the acts the controller is toperform comprise obtaining an image of a reference object with thecamera, wherein the reference object comprises a plurality of featureshaving known locations. The acts the controller is to perform furthercomprise creating a distortion map by performing acts comprisingcomparing the known locations of the plurality of features comprised bythe reference object with apparent locations of the plurality offeatures in the image of the reference object, and applying thedistortion map to each of the one or more exposures of the feature.

In some implementations of a machine such as described in any of thepreceding paragraphs of this summary, the stage is mounted on a frame ofthe machine using ball bearings, the camera is to capture images usingcomplementary metal-oxide-semiconductor sensors, and the illuminationsource is a diode laser.

In some implementations of a machine such as described in any of thepreceding paragraphs of this summary, the feature is a nanowell.

In some implementations of a machine such as described in any of thepreceding paragraphs of this summary, the analysis image is one of aplurality of analysis images; the controller is to perform a pluralityof sequencing cycles, wherein each analysis image from the plurality ofanalysis images corresponds to a single sequencing cycle; the controlleris to determine a cluster polynucleotide for each feature in the samplecontainer based on the plurality of analysis images; and the controlleris to determine a complete polynucleotide for a sample associated withthe sample container based on the cluster polynucleotides determined forthe features from the sample container.

In some implementations of a machine such as described in any of thepreceding paragraphs of this summary, the array of features comprised bythe sample container has a pitch perpendicular to the direction ofmovement of the sample container which is less than the pitch in thedirection of movement of the sample container.

In some implementations of a machine such as described in any of thepreceding paragraphs of this summary, the machine comprises a motor tocounteract movement of the stage by translating the field of view of thecamera in the direction of movement of the sample container during theperiod having the second duration.

Another implementation relates to a method comprising translating, in adirection of movement, a feature on a stage relative to a field of viewof a camera, wherein the camera has a pixel size corresponding to adistance in the direction of movement on the stage, wherein the featureis comprised by an array of features in a sample container, the array offeatures having a pitch length in the direction of movement. The methodfurther comprises, generating an analysis image by performing actscomprising, while the feature is in, and is in motion relative to, thefield of view of the camera, obtaining one or more exposures of thefeature by, for each of the one or more exposures, performing acts. Suchacts comprise exposing a sensor of the camera to illumination for afirst duration; and during a period having a second duration and whichtakes place while the sensor of the camera is exposed to illumination,illuminating the field of view of the camera with an illuminationsource. In such a method, the feature's displacement in the field ofview of the camera from beginning to end of the period having the secondduration is less than or equal to the pitch length in the direction ofmovement.

In some implementations, in a method such as described in the precedingparagraph of this summary, the feature's displacement in the directionof movement from beginning to end of the period having the secondduration is less than or equal to the pixel size.

In some implementations, in a method such as described in either of thepreceding two paragraphs of this summary obtaining one or more exposuresof the feature comprises obtaining a plurality of exposures of thefeature. The method further comprises overlaying the plurality ofexposures of the feature to create the analysis image of the feature byperforming acts comprising translating one or more of the plurality ofexposures of the feature.

In some implementations, in a method such as described in the precedingparagraph, the method comprises, for each exposure obtaining acorresponding value for the sample container's position when the fieldof view of the camera was illuminated with the illumination source; andtranslating one or more of the plurality of exposures of the featurebased on differences between the exposures' corresponding values for thesample container's position.

In some implementations, in a method such as described in the precedingparagraph, for each exposure the corresponding value for the samplecontainer's position when the field of view of the camera wasilluminated with the illumination source is obtained from an encoder.

In some implementations, in a method such as described in the precedingparagraph, the encoder has a resolution to distinguish distances smallerthan the distance on the stage corresponding to the pixel size, andoverlaying the plurality of exposures of the spot comprisesco-registering each of the plurality of exposures at the resolution ofthe encoder.

In some implementations, in a method such as described in the precedingparagraph of this summary, co-registering each of the plurality ofexposures at the resolution of the encoder comprises, for at least oneof the one or more exposures, obtaining a frequency space representationby taking a fast Fourier transform of the exposure. Co-registering eachof the plurality of exposures at the resolution of the enclosure furthercomprises, for at least one of the one or more exposures, translatingthe frequency space representation by the distance which is not a wholenumber multiple of the distance on the stage corresponding to the pixelsize; and performing an inverse fast Fourier transform of the translatedfrequency space representation.

In some implementations, in a method such as described in any of thepreceding two paragraphs, co-registering each of the plurality ofexposures comprises, for each of the plurality of exposures, upsamplingthat exposure to the resolution of the encoder based on interpolatingdata between pixels, and translating one or more of the exposures afterupsampling.

In some implementations, in a method such as described in any of thepreceding paragraphs of this summary, the sample container comprises aplurality of fiducial points, and the method comprises translating oneor more of the plurality of exposures of the feature based ondifferences in location of the fiducial points between exposures.

In some implementations, in a method such as described in any of thepreceding paragraphs of this summary, the analysis image comprises aplurality of pixels, each having a first bit depth; and each of theplurality of exposures comprises a plurality of pixels, each of whichhas a second bit depth, wherein the second bit depth is less than thefirst bit depth.

In some implementations, in a method such as described in the precedingparagraph of this summary, each pixel comprised by each image capturedby the camera has third bit depth, wherein the third bit depth isgreater than the second bit depth. Additionally, obtaining the pluralityof exposures of the feature comprises, for each exposure, capturing animage with the camera while the field of view of the camera isilluminated by the illumination source; and truncating a number of mostsignificant bits of the pixels from the image captured by the camera,wherein the truncated number of most significant bits is equal to thedifference between the third bit depth and the second bit depth.

In some implementations, in a method such as described in any of thepreceding paragraphs of this summary, a threshold illumination energydose is required for imaging the feature. Additionally, in such amethod, for each of the one or more exposures of the feature,illuminating the field of view of the camera with the illuminationsource comprises activating the illumination source at a power which,when multiplied by the second duration, provides an individual exposureenergy dose less than the threshold illumination energy dose for imagingthe feature; and, when multiplied by the second duration and multipliedby the number of exposures in the plurality of exposures, provides acollective exposure energy dose greater than the threshold illuminationenergy dose for imaging the feature.

In some implementations, in a method such as described in any of thepreceding paragraphs of this summary, the method comprises obtaining animage of a reference object with the camera, wherein the referenceobject comprises a plurality of features having known locations. Themethod may further comprise creating a distortion map by performing actscomprising comparing the known locations of the plurality of featurescomprised by the reference object with apparent locations of theplurality of features in the image of the reference object. The methodmay further comprise applying the distortion map to each of the one ormore exposures of the feature.

In some implementations, in a method such as described in any of thepreceding paragraphs of this summary, the stage is mounted on astationary frame using ball bearings, the camera is to capture imagesusing complimentary metal-oxide-semiconductor sensors, and theillumination source is a diode laser.

In some implementations, in a method such as described in any of thepreceding paragraphs of this summary, the feature is a nanowell.

In some implementations, in a method such as described in any of thepreceding paragraphs of this summary, the analysis image is one of aplurality of analysis images. In some such implementations, the methodfurther comprises performing a plurality of sequencing cycles, whereineach analysis image from the plurality of analysis images corresponds toa single sequencing cycle; determining a cluster polynucleotide for eachfeature in the sample container based on the plurality of analysisimages; and determining a complete polynucleotide for a sampleassociated with the sample container based on the clusterpolynucleotides determined for the features from the sample container.

In some implementations, in a method such as described in any of thepreceding paragraphs of this summary, the array of features comprised bythe sample container has a pitch perpendicular to the direction ofmovement of the sample container which is less than the pitch in thedirection of movement of the sample container.

In some implementations, a method such as described in any of thepreceding paragraphs of this summary, the method comprises a motorcounteracting movement of the stage by translating the field of view ofthe camera in the direction of movement during the period having thesecond duration.

Another implementation relates to a machine comprising a stage to move asample relative to a field of view of a camera which overlaps the stage.The machine further comprises the camera to capture images comprisingpixels, each of which has a pixel size corresponding to a distance onthe stage. The machine further comprises an illumination source toilluminate the field of view of the camera. The machine furthercomprises means for obtaining an analysis image of a continuously movingsample using pulsed illumination.

In some implementations, in a machine such as described in the precedingparagraph of this summary, the means for obtaining the analysis image ofthe continuously moving sample using pulsed illumination comprises meansfor translating and overlaying multiple sub-threshold exposures.

Other features and aspects of the disclosed technology will becomeapparent from the following detailed description, taken in conjunctionwith the accompanying drawings, which illustrate, by way of example, thefeatures in accordance with examples of the disclosed technology. Thesummary is not intended to limit the scope of any protection provided bythis document or any related document, which scope is defined by therespective document's claims and equivalents.

It should be appreciated that all combinations of the foregoing concepts(provided such concepts are not mutually inconsistent) are contemplatedas being part of the inventive subject matter disclosed herein. Inparticular, all combinations of claimed subject matter appearing at theend of this disclosure are contemplated as being part of the inventivesubject matter disclosed herein.

BRIEF DESCRIPTION OF THE DRAWINGS

The present disclosure, in accordance with one or more various examples,is described in detail with reference to the following figures. Thefigures are provided for purposes of illustration only and merely depicttypical or example implementations.

FIG. 1 illustrates, in one example, a generalized block diagram of anexample image scanning system with which systems and methods disclosedherein may be implemented.

FIG. 2 is block diagram illustrating an example two-channel,line-scanning modular optical imaging system that may be implemented inparticular implementations.

FIG. 3 illustrates an example configuration of a patterned sample thatmay be imaged in accordance with implementations disclosed herein.

FIG. 4 illustrates an example scenario in which a camera is used toimage a sample moving continuously through its field of view.

FIG. 5 illustrates an example process in which multiple exposures arecombined.

FIG. 6 illustrates an example computing module that may be used toimplement various features of implementations described in the presentdisclosure.

FIGS. 7A-7C illustrate configurations in which illumination from afeature is focused on a camera using lenses and a mirror.

The figures are not exhaustive and do not limit the present disclosureto the precise form disclosed.

DETAILED DESCRIPTION

As used herein to refer to a sample, the term “spot” or “feature” isintended to mean a point or area in a pattern that may be distinguishedfrom other points or areas according to relative location. An individualspot may include one or more molecules of a particular type. Forexample, a spot may include a single target nucleic acid molecule havinga particular sequence or a spot may include several nucleic acidmolecules having the same sequence (and/or complementary sequence,thereof).

As used herein to refer to a spot or feature in connection with adirection, the term “pitch” is intended to mean the separation of thespot or feature from other spots or features in the direction. Forexample, if a sample container has an array of features which areseparated from each other by 650 nm in the direction that the containerwould be moved during imaging, then the “pitch” of the features in thatdirection may be referred to as being 650 nm.

As used herein, the term “xy plane” is intended to mean a 2 dimensionalarea defined by straight line axes x and y in a Cartesian coordinatesystem. When used in reference to a detector and an object observed bythe detector, the area may be further specified as being orthogonal tothe direction of observation between the detector and object beingdetected. When used herein to refer to a line scanner, the term “ydirection” refers to the direction of scanning.

As used herein, the term “z coordinate” is intended to mean informationthat specifies the location of a point, line or area along an axes thatis orthogonal to an xy plane. In particular implementations, the z axisis orthogonal to an area of an object that is observed by a detector.For example, the direction of focus for an optical system may bespecified along the z axis.

As used herein, the term “scan a line” is intended to mean detecting a2-dimensional cross-section in an xy plane of an object, thecross-section being rectangular or oblong, and causing relative movementbetween the cross-section and the object. For example, in the case offluorescence imaging an area of an object having rectangular or oblongshape may be specifically excited (at the exclusion of other areas)and/or emission from the area may be specifically acquired (at theexclusion of other areas) at a given time point in the scan.

Implementations disclosed herein are directed to illumination of objectsto be imaged while in motion. Illumination may be provided for one ormore brief intervals, and data corresponding to multiple illuminationbrief intervals may be combined to generate an image.

FIG. 1 is an example imaging system 100 in which the technologydisclosed herein may be implemented. The example imaging system 100 mayinclude a device for obtaining or producing an image of a sample. Theexample outlined in FIG. 1 shows an example imaging configuration of abacklight design implementation. It should be noted that althoughsystems and methods may be described herein from time to time in thecontext of example imaging system 100, these are only examples withwhich implementations of the illumination and imaging techniquesdisclosed herein may be implemented.

As may be seen in the example of FIG. 1, subject samples are located onsample container 110 (e.g., a flow cell as described herein), which ispositioned on a sample stage 170 mounted on a frame 190 under anobjective lens 142. Light source 160 and associated optics direct a beamof light, such as laser light, to a chosen sample location on the samplecontainer 110. The sample fluoresces and the resultant light iscollected by the objective lens 142 and directed to an image sensor ofcamera system 140 to detect the florescence. Sample stage 170 is movedrelative to objective lens 142 to position the next sample location onsample container 110 at the focal point of the objective lens 142.Movement of sample stage 170 relative to objective lens 142 may beachieved by moving the sample stage itself, the objective lens, someother component of the imaging system, or any combination of theforegoing. Further implementations may also include moving the entireimaging system over a stationary sample.

Fluid delivery module or device 180 directs the flow of reagents (e.g.,fluorescently labeled nucleotides, buffers, enzymes, cleavage reagents,etc.) to (and through) sample container 110 and waste valve 120. Samplecontainer 110 may include one or more substrates upon which the samplesare provided. For example, in the case of a system to analyze a largenumber of different nucleic acid sequences, sample container 110 mayinclude one or more substrates on which nucleic acids to be sequencedare bound, attached or associated. In various implementations, thesubstrate may include any inert substrate or matrix to which nucleicacids may be attached, such as for example glass surfaces, plasticsurfaces, latex, dextran, polystyrene surfaces, polypropylene surfaces,polyacrylamide gels, gold surfaces, and silicon wafers. In someapplications, the substrate is within a channel or other area at aplurality of locations formed in a matrix or array across the samplecontainer 110.

In some implementations, the sample container 110 may include abiological sample that is imaged using one or more fluorescent dyes. Forexample, in a particular implementation the sample container 110 may beimplemented as a patterned flow cell including a translucent coverplate, a substrate, and a liquid sandwiched therebetween, and abiological sample may be located at an inside surface of the translucentcover plate or an inside surface of the substrate. The flow cell mayinclude a large number (e.g., thousands, millions, or billions) of wellsor other types of spots (e.g., pads, divots) that are patterned into adefined array (e.g., a hexagonal array, rectangular array, etc.) intothe substrate. Each spot may form a cluster (e.g., a monoclonal cluster)of a biological sample such as DNA, RNA, or another genomic materialwhich may be sequenced, for example, using sequencing by synthesis. Theflow cell may be further divided into a number of spaced apart lanes(e.g., eight lanes), each lane including a hexagonal array of clusters.Example flow cells that may be used in implementations disclosed hereinare described in U.S. Pat. No. 8,778,848.

The system also comprises temperature station actuator 130 andheater/cooler 135 that may optionally regulate the temperature ofconditions of the fluids within the sample container 110. Camera system140 may be included to monitor and track the sequencing of samplecontainer 110. Camera system 140 may be implemented, for example, as acharge-coupled device (CCD) camera (e.g., a time delay integration (TDI)CCD camera), which may interact with various filters within filterswitching assembly 145, objective lens 142, and focusing laser/focusinglaser assembly 150. Camera system 140 is not limited to a CCD camera andother cameras and image sensor technologies may be used. In particularimplementations, the camera sensor may have a pixel size between about 5and about 15 μm, though other pixel sizes, such as 2.4 μm may also beused in some cases.

Output data from the sensors of camera system 140 may be communicated toa real time analysis module (not shown) that may be implemented as asoftware application that analyzes the image data (e.g., image qualityscoring), reports or displays the characteristics of the laser beam(e.g., focus, shape, intensity, power, brightness, position) to agraphical user interface (GUI), and, as further described below,dynamically corrects distortion in the image data.

Light source 160 (e.g., an excitation laser within an assemblyoptionally comprising multiple lasers) or other light source may beincluded to illuminate fluorescent sequencing reactions within thesamples via illumination through a fiber optic interface (which mayoptionally comprise one or more re-imaging lenses, a fiber opticmounting, etc.). Low watt lamp 165 and focusing laser 150 also presentedin the example shown. In some implementations focusing laser 150 may beturned off during imaging. In other implementations, an alternativefocus configuration may include a second focusing camera (not shown),which may be a quadrant detector, a Position Sensitive Detector (PSD),or similar detector to measure the location of the scattered beamreflected from the surface concurrent with data collection.

Although illustrated as a backlit device, other examples may include alight from a laser or other light source that is directed through theobjective lens 142 onto the samples on sample container 110. Samplecontainer 110 may be ultimately mounted on a sample stage 170 to providemovement and alignment of the sample container 110 relative to theobjective lens 142. The sample stage may have one or more actuators toallow it to move in any of three dimensions. For example, in terms ofthe Cartesian coordinate system, actuators may be provided to allow thestage to move in the X, Y and Z directions relative to the objectivelens. This may allow one or more sample locations on sample container110 to be positioned in optical alignment with objective lens 142.

A focus (z-axis) component 175 is shown in this example as beingincluded to control positioning of the optical components relative tothe sample container 110 in the focus direction (typically referred toas the z axis, or z direction). Focus component 175 may include one ormore actuators physically coupled to the optical stage or the samplestage, or both, to move sample container 110 on sample stage 170relative to the optical components (e.g., the objective lens 142) toprovide proper focusing for the imaging operation. For example, theactuator may be physically coupled to the respective stage such as, forexample, by mechanical, magnetic, fluidic or other attachment or contactdirectly or indirectly to or with the stage. The one or more actuatorsmay move the stage in the z-direction while maintaining the sample stagein the same plane (e.g., maintaining a level or horizontal attitude,perpendicular to the optical axis). The one or more actuators may alsotilt the stage. This may be done, for example, so that sample container110 may be leveled dynamically to account for any slope in its surfaces.

Focusing of the system generally refers to aligning the focal plane ofthe objective lens with the sample to be imaged at the chosen samplelocation. However, focusing may also refer to adjustments to the systemto obtain a desired characteristic for a representation of the samplesuch as, for example, a desired level of sharpness or contrast for animage of a test sample. Because the usable depth of field of the focalplane of the objective lens may be small (sometimes on the order of 1 μmor less), focus component 175 closely follows the surface being imaged.Because the sample container is not perfectly flat as fixtured in theinstrument, focus component 175 may be set up to follow this profilewhile moving along in the scanning direction (herein referred to as they-axis).

The light emanating from a test sample at a sample location being imagedmay be directed to one or more detectors of camera system 140. Anaperture may be included and positioned to allow only light emanatingfrom the focus area to pass to the detector. The aperture may beincluded to improve image quality by filtering out components of thelight that emanate from areas that are outside of the focus area.Emission filters may be included in filter switching assembly 145, whichmay be selected to record a determined emission wavelength and to cutout any stray laser light.

Although not illustrated, a controller, which may be implemented as acomputing module such as discussed infra in the context of FIG. 6, maybe provided to control the operation of the scanning system. Thecontroller may be implemented to control aspects of system operationsuch as, for example, focusing, stage movement, and imaging operations.In various implementations, the controller may be implemented usinghardware, algorithms (e.g., machine executable instructions), or acombination of the foregoing. For example, in some implementations thecontroller may include one or more CPUs or processors with associatedmemory. As another example, the controller may comprise hardware orother circuitry to control the operation, such as a computer processorand a non-transitory computer readable medium with machine-readableinstructions stored thereon. For example, this circuitry may include oneor more of the following: field programmable gate array (FPGA),application specific integrated circuit (ASIC), programmable logicdevice (PLD), complex programmable logic device (CPLD), a programmablelogic array (PLA), programmable array logic (PAL) or other similarprocessing device or circuitry. As yet another example, the controllermay comprise a combination of this circuitry with one or moreprocessors.

Other imaging systems may also be used when implementing the disclosedtechnology. For example, FIG. 2 is block diagram illustrating an exampletwo-channel, line-scanning modular optical imaging system 200 in whichaspects of the disclosed technology may be implemented. In someimplementations, system 200 may be used for the sequencing of nucleicacids. Applicable techniques include those where nucleic acids areattached at fixed locations in an array (e.g., the wells of a flow cell)and the array is imaged repeatedly while in motion relative to the fieldof view of a camera in the imaging system 200. In such implementations,system 200 may obtain images in two different color channels, which maybe used to distinguish a particular nucleotide base type from anotherMore particularly, system 200 may implement a process referred to as“base calling,” which generally refers to a process of a determining abase call (e.g., adenine (A), cytosine (C), guanine (G), or thymine (T))for a given spot location of an image at an imaging cycle. Duringtwo-channel base calling, image data extracted from two images may beused to determine the presence of one of four base types by encodingbase identity as a combination of the intensities of the two images. Fora given spot or location in each of the two images, base identity may bedetermined based on whether the combination of signal identities is [on,on], [on, off], [off, on], or [off, off].

Referring again to imaging system 200, the system includes a linegeneration module (LGM) 210 with two light sources, 211 and 212,disposed therein. Light sources 211 and 212 may be coherent lightsources such as laser diodes which output laser beams. Light source 211may emit light in a first wavelength (e.g., a red color wavelength), andlight source 212 may emit light in a second wavelength (e.g., a greencolor wavelength). The light beams output from laser sources 211 and 212may be directed through a beam shaping lens or lenses 213. In someimplementations, a single light shaping lens may be used to shape thelight beams output from both light sources. In other implementations, aseparate beam shaping lens may be used for each light beam. In someexamples, the beam shaping lens is a Powell lens, such that the lightbeams are shaped into line patterns. The beam shaping lenses of LGM 210or other optical components imaging system may shape the light emittedby light sources 211 and 212 into a line patterns (e.g., by using one ormore Powel lenses, or other beam shaping lenses, diffractive orscattering components).

LGM 210 may further include mirror 214 and semi-reflective mirror 215 todirect the light beams through a single interface port to an emissionoptics module (EOM) 230. The light beams may pass through a shutterelement 216. EOM 230 may include objective 235 and a z-stage 236 whichmoves objective lens 235 longitudinally closer to or further away from atarget 250. For example, target 250 may include a liquid layer 252 and atranslucent cover plate 251, and a biological sample may be located atan inside surface of the translucent cover plate as well an insidesurface of the substrate layer located below the liquid layer. Thez-stage 236 may then move the objective as to focus the light beams ontoeither inside surface of the flow cell (e.g., focused on the biologicalsample). Similarly, in some implementations, the target 250 may bemounted on, or include a stage movable in the xy plane relative to theobjective lens 235. The biological sample may be DNA, RNA, proteins, orother biological materials responsive to optical sequencing as known inthe art.

EOM 230 may include semi-reflective mirror 233 to reflect a focustracking light beam emitted from a focus tracking module (FTM) 240 ontotarget 250, and then to reflect light returned from target 250 back intoFTM 240. FTM 240 may include a focus tracking optical sensor to detectcharacteristics of the returned focus tracking light beam and generate afeedback signal to optimize focus of objective 235 on target 250.

EOM 230 may also include semi-reflective mirror 234 to direct lightthrough objective lens 235, while allowing light returned from target250 to pass through. In some implementations, EOM 230 may include a tubelens 232. Light transmitted through tube lens 232 may pass throughfilter element 231 and into camera module (CAM) 220. CAM 220 may includeone or more optical sensors 221 to detect light emitted from thebiological sample in response to the incident light beams (e.g.,fluorescence in response to red and green light received from lightsources 211 and 212).

Output data from the sensors of CAM 220 may be communicated to a realtime analysis module 225. Real time analysis module, in variousimplementations, executes computer readable instructions for analyzingthe image data (e.g., image quality scoring, base calling, etc.),reporting or displaying the characteristics of the beam (e.g., focus,shape, intensity, power, brightness, position) to a graphical userinterface (GUI), etc. These operations may be performed in real-timeduring imaging cycles to minimize downstream analysis time and providereal time feedback and troubleshooting during an imaging run. Inimplementations, real time analysis module may be a computing device(e.g., computing device 1000) that is communicatively coupled to andcontrols imaging system 200. In implementations further described below,real time analysis module 225 may additionally execute computer readableinstructions for controlling illumination of the target 250 andoptionally for integrating data gathered during multiple exposures ofthe optical sensor(s) 221 into an image.

FIG. 3 illustrates an example configuration of a sample container 300that may be imaged in accordance with implementations disclosed herein.In this example, sample container 300 is patterned with a hexagonalarray of ordered spots 310 that may be simultaneously imaged during animaging run. Although a hexagonal array is illustrated in this example,in other implementations the sample container may be patterned using arectilinear array, a circular array, an octagonal array, or some otherarray pattern. For ease of illustration, sample container 300 isillustrated as having tens to hundreds of spots 310. However, as may beappreciated by one having skill in the art, sample container 300 mayhave thousands, millions, or billions of spots 310 that are imaged.Moreover, in some instances, sample container 300 may be a multi-planesample comprising multiple planes (perpendicular to focusing direction)of spots 310 that are sampled during an imaging run.

In a particular implementation, sample container 300 may be a flow cellpatterned with millions or billions of wells that are divided intolanes. In this particular implementation, each well of the flow cell maycontain biological material that is sequenced using sequencing bysynthesis.

As discussed above, illumination and imaging of an object in motionrelative to the field of view of an imaging device has been accomplishedthrough high precision motion stages, time delay integration (TDI)cameras, and diode pumped solid state lasers. However, implementationsof the disclosed technology may achieve the same goal while relaxing thenormally tight tolerances and performance requirements which are filledby those types of components. For example, in some implementations,rather than utilizing a TD camera which continuously images a samplecontainer as it moves, a different type of camera, such as a consumercamera using complimentary metal-oxide-semiconductor (CMOS) sensors maybe used to capture an image of a sample at a moment in time. In such animplementation, the operation of the implementation's light source(s)(e.g., the light source 160 from FIG. 1 or the light sources 211 212from FIG. 2) may differ from that of the light source(s) in animplementation which uses a camera that continuously images a movingtarget.

To illustrate why operations of light source(s) may be modified in animplementation with a camera which captures images of single moments intime, consider the scenario of FIG. 4, in which a camera used to image asample has a framerate that allows it to capture three exposures while afeature (e.g., nanowell) of the sample container is in its field ofview, and a resolution which allows it to split its field of view into 6pixels in the direction of movement of the sample container. In such ascenario, if the sample container was illuminated continuously while itwas in the field of view of the camera (i.e., continuously illuminatedfrom time T₁ through time T₆), the result may be two blurry images,since the exposure captured during the first frame may include photonsfrom when the feature was in the first pixel the second pixel and thethird pixel from the field of view, while the exposure captured duringthe second frame may include photons from when the feature was in thefourth pixel, the fifth pixel and the sixth pixel from the field ofview. This blurriness may result in the images being unusable. Forinstance, as shown in FIG. 4, if the sample container included threefeatures, and each of those features was separated by a distance of asingle pixel, the framerate of the camera may cause photons frommultiple features to be comingled (e.g., photons from feature 1 at T₁may be comingled with photons from feature 3 at T₃ in frame 1). This mayprevent individuals features from being distinguished from each other inthe resultant images.

A variety of measures may be taken to address blurring as describedabove. In some implementations, the distance between features on asample container in the sample container's direction of motion may beincreased, such that photons from multiple features would not becommingled given the framerate of the camera. This increase of thedistance between features in the direction of motion may be accompaniedby an increase in the spacing perpendicular to the direction of motion,or it may be made only in the direction of motion, with the spacingperpendicular to the direction of motion being left unchanged (orchanged in some other manner) If this approach were applied to thescenario of FIG. 4, such as by increasing the distance between featuresfrom one pixel to two pixels in the direction of motion of the samplecontainer, then it may be possible to distinguish individual featureseven despite blurring caused by the framerate of the camera. Similarly,in some implementations, the speed of movement of the sample containermay be decreased. For instance, if the speed of motion in the scenarioof FIG. 4 were decreased by 50%, then the individual features may bedistinguishable from each other in images taken by the camera regardlessof blurring caused by the framerate.

It may also be possible to avoid the effects of blurring by using briefperiods of illumination rather than continuous illumination of a samplecontainer. For instance, in implementations using one or more laserlight source, such as light source 160 from FIG. 1 or the light sources211 and 212 from FIG. 2, these light sources could be implemented usingpulsed lasers rather than continuous wave lasers, or the light sourcesmay be outfitted with additional components such as optical choppers toallow them to provide brief periods of illumination even if operated incontinuous wave mode. As described below, these types of approaches mayallow for a camera which captures images of a sample container atdiscrete moments to be used with a continuously moving sample containereven when a single pixel of blurriness could render the resulting imagesunusable. While the description below explains how the use of other thancontinuous illumination may allow for avoiding a pixel of blurring, thesame techniques may also be used in implementations where more than apixel of blurring is acceptable. For instance, as noted above, oneapproach to mitigating blurring may be to increase the pitch of featuresin the direction of motion of a sample container. If this type of pitchexpansion was not sufficient to avoid comingling of photons fromdifferent features (e.g., if the distance of the blurring within a framewas greater than the pitch) then the approach of expanding the pitch maybe combined with the approach of using brief periods of illumination toaddress this additional blurring. Accordingly, the discussion belowshould be understood as being illustrative of approaches to addressingblurring using brief periods of illumination, and should not be seen asimplying that that approach may only be applied where no more than apixel of blurring is acceptable.

One approach to avoid the blurring described above while imaging acontinuously moving sample container is to illuminate the samplecontainer with a pulse of sufficient intensity to allow the photonsnecessary for an image to be collected during a period which is briefenough that the distance moved by the sample container while illuminatedis less than a pixel. For example, if this type of approach were appliedto the scenario of FIG. 4, the sample container may only be illuminatedduring T₁ (or for some other period having the same or shorterduration), rather than being illuminated from T₁ through T₆ as may bethe case in an implementation using a TDI camera or similar devicedesigned to continuously image a target in motion Additionally, theintensity of illumination may be set so that the dose provided duringthe period T₁ is the same as the dose that may be provided from T₁through T₆ in an implementation using a TDI camera or similar devicedesigned to continuously image a target in motion. In this way, animplementation following this approach may avoid the blurring which mayhappen from trying to create an image from photons collected acrosspixels while still collecting sufficient photons to allow the imagecaptured by the camera to be usable for its intended purpose (e.g., toallow an imaging system such as discussed in the context of FIGS. 1 and2 to sequence a sample).

Other variations on using brief illumination to avoid blurring are alsopossible. For example, in some implementations, a sample container maybe illuminated with multiple pulses while it is in a camera's field ofview, with each pulse illuminating the sample container for a periodwhich is brief enough that the distance moved by the sample containerwhile illuminated by that pulse is less than a pixel. This may be done,for example, to avoid requiring a laser with a high enough power tocompletely illuminate a feature in less time than it takes to move adistance of one pixel, to account for saturation limits of a dye used insequencing a sample being imaged, or for other reasons as may apply in aparticular situation. In some implementations following this approach,the sample container may be illuminated once for each frame of thecamera while it is in the field of view. In this way, multiple exposuresmay be generated, with each exposure being based only on photonscollected from an illumination period too short for the sample containerto move a full pixel. To illustrate, if this approach were applied tothe scenario of FIG. 4, the sample container may be illuminated inperiods T₁ and T₄. The intensity of the illumination may also beincreased in a manner similar to that described above. That is, theintensity of illumination may be set such that the photons collectedfrom each illumination period may allow each exposure to provide ausable image.

It should be understood that the approaches described above, andexamples of how those approaches may be applied, are intended to beillustrative only, and that other approaches, and variations on thedescribed approaches, are possible and may be applied in someimplementations. To illustrate, consider the intensity of illuminationprovided in an implementation which illuminates a sample container withmultiple brief pulses while it is in a camera's field of view. In someimplementations of this type, the intensity of illumination may be setat a level which may not allow a sufficient number of photons to becollected for each exposure to provide a usable image. For example,illumination may be set at a lower intensity to reduce the risk ofphotodamage caused by repeatedly exposing a sample to high peak powerlaser illumination or to avoid reaching photo-saturation of aphosphorescent dye used in sequencing a sample. In this type ofimplementation, a process such as shown in FIG. 5 may be used to allowdata from multiple exposures to be combined to obtain (at least) oneusable image of the sample.

FIG. 5 is a flow diagram illustrating an example method 500 that may beimplemented for deriving a usable image from multiple exposures. In themethod 500 of FIG. 5, an exposure may be captured at block 501. This maybe done, for example, by exposing the sensors of a camera andilluminating a moving sample container for a brief period while it is inthe field of view of a camera, as described above in the context ofapproaches for avoiding blurring. At block 502, the position of thesample container at the time the exposure was captured may bedetermined. This may be done, for example, by, in a version where asample container was mounted on a precision motion controlled stagemoving at a constant velocity, multiplying the known velocity of thestage by the amount of time that had elapsed in the scanning processwhen the exposure was captured. This position information, along withthe exposure itself, may be stored at block 503. This process may becycled repeatedly while scanning was ongoing, with each iteration ofblocks 501 502 and 503 preferably corresponding to a single frame of thecamera being used to capture the exposures.

After scanning was complete, a method 500 such as shown in FIG. 5 maycontinue in block 504 with defining a reference position. This may bedone, for example, by defining the position of the sample container atthe time the first exposure was captured as the reference position.Then, with the reference position defined, the offset for an exposurewhich had not been processed at blocks 505-507 may be determined atblock 505. This may be done, for example, by taking a difference betweenthe position stored for the exposure being processed and the referenceposition stored previously at block 504. At block 506, the exposurebeing processed may be translated by the offset. This may be done, forexample, by adding an offset determined at block 505 to coordinates ofdata in the exposure being processed. The translated exposure may thenbe overlaid with the data from any previously processed overlays atblock 507. This may be done, for example, by doing a pixel by pixelsummation of the data from the translated exposure with the data fromany previously processed exposures, taking advantage of the fact thatthe translation may have put all exposures in a consistent coordinatesystem defined by the reference position. The operations of blocks 505,506 and 507 may then be repeated for each exposure. Once all exposureshad been overlaid, a method 500 such as shown in FIG. 5 may end at block508, and the overlay with the combined data from all of the processedexposures may treated as an image of the sample container for furtheranalysis.

Variations on, and modifications to, the method 500 of FIG. 5 are alsopossible. For example, in some implementations, rather than translatingan exposure by an offset through addition as described above in thecontext of block 506, some implementations may utilize other types oftranslation. For instance, in an implementation where the position ofthe sample container at the time an exposure is captured may bedetermined with sub-pixel accuracy, translation of an exposure such asin block 506 may be achieved by taking a Fourier transform of theexposure to be translated, multiplying the representation of theexposure in frequency space by a complex exponential defined asexp(−idk) where d is the translation amount and k is the position infrequency space, and then performing an inverse fast Fourier transformon the translated frequency space representation. Similarly, in someimplementations where the position of the sample container at the timean exposure is captured may be determined with sub-pixel accuracy, thetranslation which may be performed at block 506 may include performing alinear interpolation to determine how the sub-pixel measurement may betranslated into whole pixel measurement values in the coordinate systemdefined by the reference position. Other variations, such as scaling upexposures to have a pixel resolution matching the position resolutionand using interpolation (e.g., linear, bilinear or cubic interpolation)to fill in data for locations between pixels in the original imagebefore translation are also possible, and may be used in someimplementations. Accordingly, the examples described above of varyingtranslation approaches should be understood as being illustrative only,and should not be treated as limiting.

Variations may also be implemented to provide for optimization inrepresentation and/or processing of exposures. To illustrate, consideran implementation in which images of a sample container are captured bya 10 megapixel camera with a framerate of 1000 Hz and a bit depth of 12bits. In such a case, then the data to be processed may be generated ata rate of 120 gigabits per second. To help mitigate difficulties posedby transferring, storing and processing this amount of data, someimplementations may truncate the bit depth of the output provided by thecamera based on the amount of illumination provided for each exposure.For example, if the relationship of the framerate of the camera to thevelocity of the sample container is such that 125 exposures may becaptured of a feature of the sample container while it is in thecamera's field of view, then the illumination may be set at a levelwhich may provide each exposure 1/125 of the illumination necessary fora usable image. As a result of this lower level of illumination, none ofthe pixels from any exposures may have more than six bits of data, andso the six most significant bits of data may be truncated from eachpixel of the output of the camera before that output is processed orstored as described previously in the context of FIG. 5. Subsequently,when the exposures are overlaid in block 507, the overlaid image may beencoded at a bit depth of 12 bits per pixel, thereby reflecting allphotons captured by the camera even though none of the individualexposures were stored or encoded in a manner which may have stored thatdata. Similarly, in some implementations, some additional or alternativetypes of compression may be applied. For instance, in an implementationwhere extended sequences of bits are commonly repeated (e.g., sequencesof zeros) these sequences may be replaced by more compactrepresentations, such as through use of Huffman coding or other types ofreplacement to reduce the amount of data required to represent therelevant data.

Another type of variation which may be included in some implementationsis to add additional processing acts to further account for requirementsof components that may be used. For example, in some implementations, acamera may capture images of a sample container using high precision lowdistortion optics. However, in other implementations, rather than usinglow distortion optics, additional processing acts may be performed toaccount for imperfections that may be introduced by capturing exposuresusing a camera with a less precisely manufactured lens. For instance, insome implementations, prior to using a method such as shown in FIG. 5 toimage a sample container, a calibration process may be performed inwhich the method of FIG. 5 may be used to image a calibration targetlithographically patterned with a plurality of holes in known aconfiguration (e.g., the pattern of features 310 illustrated in FIG. 3).The pattern of holes in the image of the target captured by the cameramay then be compared with the known pattern of holes, and a polynomialmap representing the warping introduced by imperfections in the camera'slens may be created from this comparison. Subsequently, when the camerawas used in imaging the sample container, this polynomial map may beapplied to reverse the distortion introduced by the lenses in theimage(s) of the sample container, thereby allowing the tolerancesnormally required for low distortion optics in a system such as shown inFIG. 1 or 2 to be relaxed in some implementations. As another example,in some implementations, a light source may illuminate a samplecontainer with multiple pulses before the distance moved by the samplecontainer reached the size of a pixel in the camera capturing images ofthe same. This may be used, for instance, if a light source was not ableto provide sufficiently intense illumination continuously during thetime needed for the sample container to move the distance of one pixel,but may provide multiple higher intensity, but briefer, pulses duringthat time.

Additional components may also, or alternatively, be included in someimplementations to address and/or mitigate the constraints provided bydiscontinuous illumination. For example, in some cases, imagestabilization techniques may be used to make the sample container appearstationary in the camera's field of view, thereby reducing the impact ofthe container's movement and potentially increasing the amount of timethat the container could be illuminated during any frame. This may bedone, for instance, by using a motor to shift the camera (or a lens ofthe camera) in a manner which is synchronized with the movement of thestage, thereby moving the camera's field of view during a frame suchthat the sample container would appear stationary (or to move a distanceof less than one pixel). Alternatively, in some cases which use thistype of image stabilization approach, a piezo or galvo mirror may beplaced in the path of the emissions from the sample container, again,effectively allowing the camera's field of view to be moved in a mannerthat counteract the movement of the stage during the portion of a framewhen the sample container was illuminated. When the sample container wasno longer illuminated, the motor could reset the field of view for thenext frame, and this process could be repeated for the duration of animaging run.

To illustrate a potential implementation of how image stabilizationcould potentially be used to mitigate the constraints provided bydiscontinuous illumination, consider FIGS. 7A-7C In those figures, FIG.7A illustrates a relationship of a feature 701, a camera 702 whose fieldof view is split into 12 pixels, and a movable mirror (e.g., a galvomirror) 703 used for image stabilization. In that figure, when light isemitted from the feature, it would be directed to the mirror 703 by afirst lens 704 reflected off the mirror 703 to a second lens 705, andfocused by the second lens 705 onto a first pixel of the camera 702where it would be detected. FIG. 7B illustrates a result of the movementof the feature 701 over a distance greater than or equal to the size ofa pixel on the camera 702 if the feature had been continuouslyilluminated, the mirror 703 had remained stationary, and the camera 702had captured only a single exposure during the period of that movement.As shown in FIG. 7B, this may result in the signal from the feature 701being spread across multiple pixels on the camera 702, and couldpotentially result in signal overlap in the event that a second feature(not shown in FIG. 7B) had been adjacent to the feature 701 shown inFIGS. 7A and 7B. By contrast, FIG. 7C shows a result of moving themirror 703 while the feature 701 moved a distance greater than one pixelon the camera 702. As shown in FIG. 7C, by moving the mirror 703 tocompensate for the movement of the feature 701, the light emitted fromthe feature could be continuously focused on a single pixel, therebyavoiding the blurring shown in FIG. 7B. As described previously this mayalso be achieved via movement of the camera 702, or a lens used todirect or focus illumination from the feature (e.g., the first lens 704or the second lens 705 illustrated in FIGS. 7A-7C). Accordingly, thespecific configuration and components shown in FIGS. 7A-7C should beunderstood as being illustrative only, and should not be treated aslimiting.

While the above examples and discussion focused on variations onillumination and image capture components, it should be understood thatvariations in other types of components may be used in someimplementations as well. To illustrate, consider stages for moving asample container through a field of view of an imaging device. In someimplementations, the stage may be implemented with components such ascross roller bearings to enable its motion to be precisely controlled(e.g., implementations which determine the position of the samplecontainer when an exposure is captured based on assumptions regardingthe uniformity of the stage's motion). However, in otherimplementations, a stage with less precise motion control may be used,such as a friction based stage or one mounted on the frame of an imagingsystem with ball bearings, and an additional component, such as anencoder, may be introduced to determine the location of the stage at thespecific points in time when exposures of a sample container arecaptured. In such an implementation, determining the position of anexposure such as illustrated in block 502 of FIG. 5 may be performed byquerying the encoder for the position of the stage when the samplecontainer is illuminated for an exposure, rather than by determining theposition based on time. Alternative position determination features mayalso be used. For instance, in some variations, a sample container maybe instrumented with a set of bright beads which could operate asfiducial reference points to allow relative positions of the samplecontainer captured in different images to be determined so that featuresin those images could be co-registered with each other as describedabove.

Of course, variations on this may also be possible as well. For example,in some implementations, an exposure may be stored with timeinformation, rather than with its position as described in block 503. Inthis type of implementation, the actual position of the exposure mayonly be determined subsequently when it is necessary to calculate anoffset, such as by multiplying by known movement speed as describedpreviously in the context of block 502, or by matching the time for theexposure against timestamped location information collected from anencoder during scanning. Other variations, such as capturing multiplelocations per illumination pulse (e.g., at the beginning and end of thepulse) and then averaging them to obtain a location for the pulse'scorresponding exposure, or omitting a position determination anddetermining exposure offsets by comparing locations of fiducialreference points may also be possible in some implementations.Accordingly, the examples provided above should be understood as beingillustrative only, and should not be treated as limiting.

Some implementations may also feature methods which vary from theoverall structure of the method of FIG. 5. For example, in someimplementations, rather than capturing and storing multiple exposuresand then overlaying the previously captured and stored exposures, thedetermining of offsets, translation of offsets, and the overlaying oftranslated exposures may be performed in real time on an exposure byexposure basis as each exposure is captured. In such an implementation,acts such as described previously in the context of blocks 501, 502,504, 505, 506 and 507 may be repeatedly performed while scanningproceeds, potentially allowing a user to see the progress of imaging asample as exposures are captured.

To further illustrate how aspects of the disclosed technology may beapplied in practice, consider a scenario in which a biological sample issplit into clusters in nanowells in an array having a pitch length inthe direction of movement of 624 nm, and the data captured from thenanowells is to be used for DNA sequencing using sequencing bysynthesis. In such a case, if the sample container is to be imaged whilemoving at 10 mm/s through a 1×1 mm field of view of a 1000 Hz camera,and each pixel in the camera corresponds to a distance of 0.3 μm in thefield of view, an implementation using a method such as shown in FIG. 5may capture 100 exposures of each nanowell while it was in the camera'sfield of view for each sequencing cycle, based on the framerate of thecamera, the field of view of the camera, and the speed of movement ofthe sample container (i.e., exposures=framerate of camera*length offield of view/speed of movement). Additionally, in such animplementation, the sample container may be illuminated for 0.03 ms orless per exposure, based on the size of the camera's pixels, themovement speed of the sample and the framerate of the camera (i.e.,illumination time:=(pixel size/speed of movement)*framerate). In such ascenario, if a threshold dose on the order of 1-5 J/cm² is necessary toimage each nanowell correctly, then an implementation following themethod of FIG. 5 may obtain a usable image of the sample for eachsequencing cycle by illuminating the sample using a laser havingcontinuous wave power in the range of 3.3-16.5 W in combination with anoptical chopper to control the duration of illumination, based on therequired dose, field of view (FOV), and the duration of eachillumination pulse (i.e., power=dose*FOV area/pulse duration). This maybe provided in a variety of ways, including diode pumped solid state(DPSS) lasers as described previously, or using less expensivecomponents, such as diode lasers. These images may then be used toidentify the sequences of nucleotides in the cluster in each nanowell,and those clusters may then be combined as they may be in preexistingsequencing by synthesis such as may be performed using data gatheredusing continuous illumination of a sample.

FIG. 6 illustrates an example computing component that may be used toimplement various features of the system and methods disclosed herein,such as the aforementioned features and functionality of one or moreaspects of methods 400 and 450. For example, computing component may beimplemented as a real-time analysis module 225.

As used herein, the term module may describe a given unit offunctionality that may be performed in accordance with one or moreimplementations of the present application. As used herein, a module maybe implemented utilizing any form of hardware, software, or acombination thereof. For example, one or more processors, controllers,ASICs, PLAs, PALs, CPLDs, FPGAs, logical components, software routinesor other mechanisms may be implemented to make up a module. Inimplementation, the various modules described herein may be implementedas discrete modules or the functions and features described may beshared in part or in total among one or more modules. In other words, asmay be apparent to one of ordinary skill in the art after reading thisdescription, the various features and functionality described herein maybe implemented in any given application and may be implemented in one ormore separate or shared modules in various combinations and permutationsEven though various features or elements of functionality may beindividually described or claimed as separate modules, one of ordinaryskill in the art will understand that these features and functionalitymay be shared among one or more common software and hardware elements,and such description shall not require or imply that separate hardwareor software components are used to implement such features orfunctionality.

Where components or modules of the application are implemented in wholeor in part using software, in one implementation, these softwareelements may be implemented to operate with a computing or processingmodule capable of carrying out the functionality described with respectthereto. One such example computing module is shown in FIG. 6. Variousimplementations are described in terms of this example-computing module1000. After reading this description, it will become apparent to aperson skilled in the relevant art how to implement the applicationusing other computing modules or architectures.

Referring now to FIG. 6, computing module 1000 may represent, forexample, computing or processing capabilities found within desktop,laptop, notebook, and tablet computers, hand-held computing devices(tablets, PDA's, smart phones, cell phones, palmtops, etc.); mainframes,supercomputers, workstations or servers; or any other type ofspecial-purpose or general-purpose computing devices as may be desirableor appropriate for a given application or environment. Computing module1000 may also represent computing capabilities embedded within orotherwise available to a given device. For example, a computing modulemay be found in other electronic devices such as, for example, digitalcameras, navigation systems, cellular telephones, portable computingdevices, modems, routers, WAPs, terminals and other electronic devicesthat may include some form of processing capability.

Computing module 1000 may include, for example, one or more processors,controllers, control modules, or other processing devices, such as aprocessor 1004. Processor 1004 may be implemented using ageneral-purpose or special-purpose processing engine such as, forexample, a microprocessor, controller, or other control logic. In theillustrated example, processor 1004 is connected to a bus 1002, althoughany communication medium may be used to facilitate interaction withother components of computing module 1000 or to communicate externally.

Computing module 1000 may also include one or more memory modules,referred to herein as main memory 1008. For example, preferably randomaccess memory (RAM) or other dynamic memory, may be used for storinginformation and instructions to be executed by processor 1004. Mainmemory 1008 may also be used for storing temporary variables or otherintermediate information during execution of instructions to be executedby processor 1004. Computing module 1000 may likewise include a readonly memory (“ROM”) or other static storage device coupled to bus 1002for storing static information and instructions for processor 1004.

The computing module 1000 may also include one or more various forms ofinformation storage mechanism 1010, which may include, for example, amedia drive 1012 and a storage unit interface 1020. The media drive 1012may include a drive or other mechanism to support fixed or removablestorage media 1014. For example, a hard disk drive, a solid state drive,a magnetic tape drive, an optical disk drive, a CD or DVD drive (R orRW), or other removable or fixed media drive may be provided.Accordingly, storage media 1014 may include, for example, a hard disk, asolid state drive, magnetic tape, cartridge, optical disk, a CD, DVD, orBlu-ray, or other fixed or removable medium that is read by, written toor accessed by media drive 1012. As these examples illustrate, thestorage media 1014 may include a computer usable storage medium havingstored therein computer software or data.

In alternative implementations, information storage mechanism 1010 mayinclude other similar instrumentalities for allowing computer programsor other instructions or data to be loaded into computing module 1000.Such instrumentalities may include, for example, a fixed or removablestorage unit 1022 and an interface 1020. Examples of such storage units1022 and interfaces 1020 may include a program cartridge and cartridgeinterface, a removable memory (for example, a flash memory or otherremovable memory module) and memory slot, a PCMCIA slot and card, andother fixed or removable storage units 1022 and interfaces 1020 thatallow software and data to be transferred from the storage unit 1022 tocomputing module 1000.

Computing module 1000 may also include a communications interface 1024.Communications interface 1024 may be used to allow software and data tobe transferred between computing module 1000 and external devices.Examples of communications interface 1024 may include a modem orsoftmodem, a network interface (such as an Ethernet, network interfacecard, WiMedia, IEEE 802.XX or other interface), a communications port(such as for example, a USB port, IR port, RS232 port Bluetooth®interface, or other port), or other communications interface. Softwareand data transferred via communications interface 1024 may typically becarried on signals, which may be electronic, electromagnetic (whichincludes optical) or other signals capable of being exchanged by a givencommunications interface 1024. These signals may be provided tocommunications interface 1024 via a channel 1028. This channel 1028 maycarry signals and may be implemented using a wired or wirelesscommunication medium. Some examples of a channel may include a phoneline, a cellular link, an RF link, an optical link, a network interface,a local or wide area network, and other wired or wireless communicationschannels.

In this document, the terms “computer readable medium”, “computer usablemedium” and “computer program medium” are used to generally refer tonon-transitory media, volatile or non-volatile, such as, for example,memory 1008, storage unit 1022, and media 1014. These and other variousforms of computer program media or computer usable media may be involvedin carrying one or more sequences of one or more instructions to aprocessing device for execution. Such instructions embodied on themedium, are generally referred to as “computer program code” or a“computer program product” (which may be grouped in the form of computerprograms or other groupings) When executed, such instructions may enablethe computing module 1000 to perform features or functions of thepresent application as discussed herein.

In the claims, the phrase “means for obtaining an analysis image of acontinuously moving sample using pulsed illumination” should beunderstood as a means plus function limitation as provided for in 35U.S.C. § 112(f) in which the function is obtaining an analysis image ofa continuously moving sample using pulsed illumination, and thecorresponding structure is an illumination source, a camera, a movingstage, and a computer as described in the context of FIG. 4 to causesub-pixel illumination pulses and otherwise avoid blurring as may becaused by continuous illumination.

In the claims, the phrase “means for translating and overlaying multiplesub-threshold exposures” should be understood as a means plus functionlimitations as provided for in 35 U.S.C. § 112(t) in which the functionis “translating and overlaying multiple sub-threshold exposures” and thecorresponding structure is a computer to perform acts as described inthe context of blocks 505-507 of FIG. 5, as well as the variations onthose acts described above as being included in some implementations.

Although described above in terms of various implementations andimplementations, it should be understood that the various features,aspects and functionality described in one or more of the individualimplementations are not limited in their applicability to the particularimplementation with which they are described, but instead may beapplied, alone or in various combinations, to one or more of the otherimplementations of the application, whether or not such implementationsare described and whether or not such features are presented as being apart of a described implementation. Thus, the breadth and scope ofprotection provided by this document or any related document should notbe limited by any of the above-described implementations.

It should be appreciated that all combinations of the foregoing concepts(provided such concepts are not mutually inconsistent) are contemplatedas being part of the inventive subject matter disclosed herein. Inparticular, all combinations of claimed subject matter appearing at theend of this disclosure are contemplated as being part of the inventivesubject matter disclosed herein.

The terms “substantially” and “about” used throughout this disclosure,including the claims, are used to describe and account for smallfluctuations, such as due to variations in processing. For example, theymay refer to less than or equal to 5%, such as less than or equal to±2%, such as less than or equal to ±1%, such as less than or equal to±0.5%, such as less than or equal to ±0.2%, such as less than or equalto ±0.1%, such as less than or equal to ±0.05%.

To the extent applicable, the terms “first.” “second,” “third,” etc.herein are merely employed to show the respective objects described bythese terms as separate entities and are not meant to connote a sense ofchronological order, unless stated explicitly otherwise herein.

Terms and phrases used in this document, and variations thereof, unlessotherwise expressly stated, should be construed as open ended as opposedto limiting. As examples of the foregoing: the term “including” shouldbe read as meaning “including, without limitation” or the like; the term“example” is used to provide instances of the item in discussion, not anexhaustive or limiting list thereof; the terms “a” or “an” should beread as meaning “at least one,” “one or more” or the like; andadjectives such as “preexisting,” “traditional,” “normal,” “standard,”“known” and terms of similar meaning should not be construed as limitingthe item described to a given time period or to an item available as ofa given time, but instead should be read to encompass preexisting,traditional, normal, or standard technologies that may be available orknown now or at any time in the future. Likewise, where this documentrefers to technologies that may be apparent or known to one of ordinaryskill in the art, such technologies encompass those apparent or known tothe skilled artisan now or at any time in the future.

The presence of broadening words and phrases such as “one or more,” “atleast,” “but not limited to” or other like phrases in some instancesshall not be read to mean that the narrower case is intended or requiredin instances where such broadening phrases may be absent. The use of theterm “module” does not imply that the components or functionalitydescribed or claimed as part of the module are all configured in acommon package. Indeed, any or all of the various components of amodule, whether control logic or other components, may be combined in asingle package or separately maintained and may further be distributedin multiple groupings or packages or across multiple locations.

Additionally, the various implementations set forth herein are describedin terms of block diagrams, flow charts and other illustrations. As willbecome apparent to one of ordinary skill in the art after reading thisdocument, the illustrated implementations and their various alternativesmay be implemented without confinement to the illustrated examples. Forexample, block diagrams and their accompanying description should not beconstrued as mandating a particular architecture or configuration.

While various implementations of the present disclosure have beendescribed above, it should be understood that they have been presentedby way of example only, and not of limitation. Likewise, the variousdiagrams may depict an example architectural or other configuration forthe disclosure, which is done to aid in understanding the features andfunctionality that may be included in the disclosure. The disclosure isnot restricted to the illustrated example architectures orconfigurations, but the desired features may be implemented using avariety of alternative architectures and configurations. Indeed, it willbe apparent to one of skill in the art how alternative functional,logical or physical partitioning and configurations may be implementedto implement the desired features of the present disclosure. Also, amultitude of different constituent module names other than thosedepicted herein may be applied to the various partitions. Additionally,with regard to flow diagrams, operational descriptions and methodclaims, the order in which the acts are presented herein shall notmandate that various implementations be implemented to perform therecited functionality in the same order unless the context dictatesotherwise.

What is claimed is:
 1. A machine comprising: a camera to capture imagescomprising pixels, each of which has a pixel size corresponding to adistance on a stage in a direction of movement of a sample container;the stage to move the sample container relative to a field of view ofthe camera which overlaps the stage, wherein the sample containercomprises an array of features having a pitch length in a direction ofmovement of the sample container; an illumination source to illuminatethe field of view of the camera; and a controller to obtain an analysisimage by performing acts comprising, while a feature from the array offeatures is in, and is in motion relative to, the field of view of thecamera, obtaining one or more exposures of the feature by, for each ofthe one or more exposures, performing acts comprising: exposing a sensorof the camera to illumination for a first duration; and during a periodhaving a second duration and which takes place while the sensor of thecamera is exposed to illumination, illuminating the field of view of thecamera with the illumination source; wherein the feature's displacementin the field of view of the camera from beginning to end of the periodhaving the second duration is less than or equal to the pitch length inthe direction of movement of the sample container.
 2. The machine ofclaim 1, wherein the feature's displacement in the direction of movementof the sample container in the field of view of the camera frombeginning to end of the period having the second duration is less thanor equal to the pixel size.
 3. The machine of claim 1, wherein:obtaining one or more exposures of the feature comprises obtaining aplurality of exposures of the feature; the acts the controller is toperform comprise overlaying the plurality of exposures of the featurebased on translating one or more of the plurality of exposures of thefeature.
 4. The machine of claim 3, wherein: the analysis imagecomprises a plurality of pixels, each having a first bit depth; and eachof the plurality of exposures comprises a plurality of pixels, each ofwhich has a second bit depth, wherein the second bit depth is less thanthe first bit depth.
 5. The machine of claim 4, wherein: each pixelcomprised by each image captured by the camera has third bit depth,wherein the third bit depth is greater than the second bit depth;obtaining the plurality of exposures of the feature comprises, for eachexposure: capturing an image with the camera while the field of view ofthe camera is illuminated by the illumination source; and truncating anumber of most significant bits of the pixels from the image captured bythe camera, wherein the truncated number of most significant bits isequal to the difference between the third bit depth and the second bitdepth.
 6. The machine of claim 1, wherein: a threshold illuminationenergy dose is required for imaging the feature; for each of the one ormore exposures of the feature, illuminating the field of view of thecamera with the illumination source comprises activating theillumination source at a power which: when multiplied by the secondduration, provides an individual exposure energy dose less than thethreshold illumination energy dose for imaging the feature; and whenmultiplied by the second duration and multiplied by the number ofexposures in the plurality of exposures of the feature, provides acollective exposure energy dose greater than the threshold illuminationenergy dose for imaging the feature.
 7. The machine of claim 1, wherein:the stage is mounted on a frame of the machine using ball bearings; thecamera is to capture images using complementarymetal-oxide-semiconductor sensors; and the illumination source is adiode laser.
 8. The machine of claim 1, wherein: the analysis image isone of a plurality of analysis images; the controller is to perform aplurality of sequencing cycles, wherein each analysis image from theplurality of analysis images corresponds to a single sequencing cycle;and the controller is to determine a cluster polynucleotide for eachfeature in the sample container based on the plurality of analysisimages; and the controller is to determine a complete polynucleotide fora sample associated with the sample container based on the clusterpolynucleotides determined for the features from the sample container.9. The machine of claim 1, wherein the machine comprises a motor tocounteract movement of the stage by translating the field of view of thecamera in the direction of movement of the sample container during theperiod having the second duration.
 10. A method comprising: translating,in a direction of movement, a feature on a stage relative to a field ofview of a camera, wherein the camera has a pixel size corresponding to adistance in the direction of movement on the stage, wherein the featureis comprised by an array of features in a sample container, the array offeatures having a pitch length in the direction of movement; generatingan analysis image by performing acts comprising, while the feature isin, and is in motion relative to, the field of view of the camera,obtaining one or more exposures of the feature by, for each of the oneor more exposures, performing acts comprising: exposing a sensor of thecamera to illumination for a first duration; during a period having asecond duration and which takes place while the sensor of the camera isexposed to illumination, illuminating the field of view of the camerawith an illumination source; wherein the feature's displacement of thefield of view of the camera from beginning to end of the period havingthe second duration is less than or equal to the pitch length in thedirection of movement.
 11. The method of claim 10, wherein the feature'sdisplacement in the direction of movement from beginning to end of theperiod having the second duration is less than or equal to the pixelsize.
 12. The method of claim 9, wherein: obtaining one or moreexposures of the feature comprises obtaining a plurality of exposures ofthe feature; the method comprises overlaying the plurality of exposuresof the feature to create the analysis image of the feature by performingacts comprising translating one or more of the plurality of exposures ofthe feature.
 13. The method of claim 12, wherein: the analysis imagecomprises a plurality of pixels, each having a first bit depth; and eachof the plurality of exposures comprises a plurality of pixels, each ofwhich has a second bit depth, wherein the second bit depth is less thanthe first bit depth.
 14. The method of claim 13, wherein: each pixelcomprised by each image captured by the camera has third bit depth,wherein the third bit depth is greater than the second bit depth;obtaining the plurality of exposures of the feature comprises, for eachexposure: capturing an image with the camera while the field of view ofthe camera is illuminated by the illumination source; and truncating anumber of most significant bits of the pixels from the image captured bythe camera, wherein the truncated number of most significant bits isequal to the difference between the third bit depth and the second bitdepth.
 15. The method of claim 10, wherein: a threshold illuminationenergy dose is required for imaging the feature; for each of the one ormore exposures of the feature, illuminating the field of view of thecamera with the illumination source comprises activating theillumination source at a power which: when multiplied by the secondduration, provides an individual exposure energy dose less than thethreshold illumination energy dose for imaging the feature; and whenmultiplied by the second duration and multiplied by the number ofexposures in the plurality of exposures, provides a collective exposureenergy dose greater than the threshold illumination energy dose forimaging the feature.
 16. The method of claim 10, wherein: the stage ismounted on a stationary frame using ball bearings; the camera is tocapture images using complimentary metal-oxide-semiconductor sensors;and the illumination source is a diode laser.
 17. The method of claim10, wherein: the analysis image is one of a plurality of analysisimages; the method comprises: performing a plurality of sequencingcycles, wherein each analysis image from the plurality of analysisimages corresponds to a single sequencing cycle; determining a clusterpolynucleotide for each feature in the sample container based on theplurality of analysis images; and determining a complete polynucleotidefor a sample associated with the sample container based on the clusterpolynucleotides determined for the features from the sample container.18. The method of claim 10, wherein the method comprises a motorcounteracting movement of the stage by translating the field of view ofthe camera in the direction of movement during the period having thesecond duration.
 19. A machine comprising: a stage to move a samplerelative to a field of view of a camera which overlaps the stage; thecamera to capture images comprising pixels, each of which has a pixelsize corresponding to a distance on the stage; an illumination source toilluminate the field of view of the camera; and means for obtaining ananalysis image of a continuously moving sample using pulsedillumination.
 20. The machine of claim 19, wherein the means forobtaining the analysis image of the continuously moving sample usingpulsed illumination comprises means for translating and overlayingmultiple sub-threshold exposures.